Tobacco specific nitrosamines (TSNAs) are formed primarily during the curing and processing of tobacco leaves. Tobacco curing is a process of physical and biochemical changes that bring out the aroma and flavor of each variety of tobacco. It is believed that the amount TSNA in cured tobacco leaf is dependent on the accumulation of nitrites, which accumulate during the death of the plant cell and are formed during curing by the reduction of nitrates under conditions approaching an anaerobic (oxygen deficient) environment. The reduction of nitrates to nitrites is believed to occur by the action of bacteria on the surface of the leaf under anaerobic conditions, and this reduction is particularly pronounced under certain conditions. Once nitrites are formed, these compounds are believed to combine with various tobacco alkaloids, including pyridine-containing compounds, to form nitrosamines.
The four principal TSNAs, that is, those typically found to be present in the highest concentrations, are N-nitrosonicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). Minor compounds, that is, those typically found at significantly lower levels than the principal TSNAs, include 4-(methylnitrosamino) 4-(3-pyridyl) butanal (NNA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), 4-(methylnitrosamino)4-(3-pyridyl)-1-butanol (iso-NNAL), and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butyric acid (iso-NNAC). At least NNN and NNK have been reported to be carcinogenic when applied to animals in laboratory studies.
The primary biochemical mechanism of NNN formation is the N-nitrosation of nornicotine, an alkaloid produced through the N-demethylation of nicotine by the enzyme nicotine N-demethylase. Although nornicotine typically represents <5% of the total alkaloid content in cultivated tobacco, nornicotine levels can dramatically increase by a mechanism termed “conversion” in which plants that accumulate nicotine as their principal alkaloid give rise to progeny that metabolize a large portion (as high as 95%) of leaf nicotine to nornicotine. In individuals that have genetically converted (termed “converters”), N-demethylation of nicotine to nornicotine primarily occurs during senescence and curing. Maintaining low nornicotine levels is desirable because of its well characterised role as the precursor of NNN and also because nornicotine per se may be responsible for unwanted health effects. Dickerson and Janda (2002) Proc. Natl. Acad. Sci. USA 99, 15084-15088 demonstrated that nornicotine can induce aberrant glycation of proteins and showed the increased accumulation of modified proteins in the blood plasma of smokers. Furthermore, the same report provided evidence that nornicotine can react covalently with commonly used steroid drugs, such as prednisone, potentially altering both the efficacy and toxicity of these drugs. WO98/58555 describes the treatment of tobacco leaves before or during flue-curing by microwaving for reducing TSNAs. U.S. Pat. No. 5,810,020 describes a process for removing TSNAs from tobacco by contacting the tobacco material with a trapping sink, wherein the trapping sink comprises a select transition metal complex which is readily nitrosated to form a nitrosyl complex with little kinetic or thermodynamic hindrance. U.S. Pat. No. 6,202,649 describes a method of substantially preventing formation of TSNAs by, among other things, curing tobacco in a controlled environment having a sufficient airflow to substantially prevent an anaerobic condition around the vicinity of the tobacco leaf. The controlled environment is provided by controlling one or more curing parameters, such as airflow, humidity, and temperature. However, methods such as these can add considerable cost and time to the production of tobacco and therefore are less likely to be accepted by the tobacco industry. Thus, a need remains for an effective and relatively inexpensive method for reducing TSNAs. Molecular based methods for reducing the levels of TSNAs in plants are highly desirable since they do not require expensive, and often complex, methods to achieve the reduced levels of TSNAs. One such molecular based approach is disclosed in WO2011/088180. Compositions and methods are disclosed in WO2011/088180 for inhibiting the expression or function of a root-specific nicotine demethylase polypeptide (CYP82E10) that is involved in the metabolic conversion of nicotine to nornicotine in the roots of tobacco plants. Nicotine demethylase belongs to the family of the cytochrome P450 monooxygenases (CYP). Other nicotine demethylase genes have been described including CYP82E4 and CYP82E5 which participate in the conversion of nicotine to nornicotine and are described in WO2006091194, WO2008070274 and WO2009064771. The knockout of CYP82E4, CYP82E5 and CYP82E10 is able to reduce conversion of nicotine to nornicotine from 3.2% down to 1.1% in Burley tobacco (see WO 2011088180 A1).
There is a continuing need in the art to further reduce the levels of nornicotine in tobacco plants to further reduce the levels of metabolities of nornicotine (for example, TSNAs—such as NNN) that are formed during curing. The present invention seeks to address this need.